Intraspinal botulinum toxin for treating pain

ABSTRACT

Methods for treating pain by intrathecal administration to a human patient of a therapeutically effective amount of a neurotoxin such as botulinum toxin type A are disclosed.

BACKGROUND

[0001] The present invention relates to methods for treating pain. Inparticular, the present invention relates to methods for treating painby intraspinal administration of a neurotoxin.

[0002] Many, if not most ailments of the body cause pain. Generally painis experienced when the free nerve endings which constitute the painreceptors in the skin as well as in certain internal tissues aresubjected to mechanical, thermal or chemical stimuli. The pain receptorstransmit signals along afferent neurons into the central nervous systemand thence to the brain.

[0003] The causes of pain can include inflammation, injury, disease,muscle spasm and the onset of a neuropathic event or syndrome.Ineffectively treated pain can be devastating to the person experiencingit by limiting function, reducing mobility, complicating sleep, anddramatically interfering with the quality of life.

[0004] Inflammatory pain can occur when tissue is damaged, as can resultfrom surgery or due to an adverse physical, chemical or thermal event orto infection by a biologic agent. Although inflammatory pain isgenerally reversible and subsides when the injured tissue has beenrepaired or the pain inducing stimulus removed, present methods fortreating inflammatory pain have many drawbacks and deficiencies. Thus,the typical oral, parenteral or topical administration of an analgesicdrug to treat the symptoms of pain or of, for example, an antibiotic totreat inflammatory pain causation factors can result in widespreadsystemic distribution of the drug and undesirable side effects.Additionally, current therapy for inflammatory pain suffers from shortdrug efficacy durations which necessitate frequent drug readministrationwith possible resulting drug resistance, antibody development and/ordrug dependence and addiction, all of which are unsatisfactory.Furthermore, frequent drug administration increases the expense of theregimen to the patient and can require the patient to remember to adhereto a dosing schedule.,

[0005] Neuropathic pain is a persistent or chronic pain syndrome thatcan result from damage to the nervous system, the peripheral nerves, thedorsal root ganglion or dorsal root, or to the central nervous system.Neuropathic pain syndromes include allodynia, various neuralgias such aspost herpetic neuralgia and trigeminal neuralgia, phantom pain, andcomplex regional pain syndromes, such as reflex sympathetic dystrophyand causalgia. Causalgia is characterized by spontaneous burning paincombined with hyperalgesia and allodynia.

[0006] Unfortunately, current methods to treat neuropathic pain, such asby local anesthetic blocks targeted to trigger points, peripheralnerves, plexi, dorsal roots, and to the sympathetic nervous system haveonly short-lived antinociceptive effects. Additionally, longer lastinganalgesic treatment methods, such as blocks by phenol injection orcryotherapy raise a considerable risk of irreversible functionalimpairment. Furthermore, chronic epidural or intrathecal (collectively“intraspinal”) administration of drugs such as clonidine, steroids,opioids or midazolam have significant side effects and questionableefficacy.

[0007] Tragically there is no existing method for adequately,predictably and specifically treating established neuropathic pain(Woolf C. et al., Neuropathic Pain: Aetiology, Symptoms, Mechanisms, andManagement, Lancet 1999; 353: 1959-64) as present treatment methods forneuropathic pain consists of merely trying to help the patient copethrough psychological or occupational therapy, rather than by reducingor eliminating the pain experienced.

[0008] Spasticity or muscle spasm can be a serious complication oftrauma to the spinal cord or other disorders that create damage withinthe spinal cord and the muscle spasm is often accompanied by pain. Thepain experienced during a muscle spasm can result from the direct effectof the muscle spasm stimulating mechanosensitive pain receptors or fromthe indirect effect of the spasm compressing blood vessels and causingischemia. Since the spasm increases the rate of metabolism in theaffected muscle tissue, the relative ischemia becomes greater creatingthereby conditions for the release of pain inducing substances.

[0009] Within the enclosure by the vertebral canal for the spinal cordby the bones of the vertebrae, the spinal cord is surrounded by threemeningeal sheaths which are continuous with those which encapsulate thebrain. The outermost of these three meningeal sheaths is the duramatter, a dense, fibrous membrane which anteriorally is separated fromthe periosteum of the vertebral by the epidural space. Posterior to thedura matter is the subdural space. The subdural space surrounds thesecond of the three meningeal sheaths which surround the spinal cord,the arachnoid membrane. The arachnoid membrane is separated from thethird meningeal sheath, the pia mater, by the subarachnoid orintrathecal space. The subarachnoid space is filled with cerebrospinalfluid (CSF). Underlying the pia mater is the spinal cord. Thus theprogression proceeding inwards or in posterior manner from the vertebrais the epidural space, dura mater, subdural space, arachnoid membrane,intrathecal space, pia matter and spinal cord.

[0010] Therapeutic administration of certain drugs intraspinally, thatis to either the epidural space or to the intrathecal space, is known.Administration of a drug directly to the intrathecal space can be byeither spinal tap injection or by catheterization. Intrathecal drugadministration can avoid the inactivation of some drugs when takenorally as well and the systemic effects of oral or intravenousadministration. Additionally, intrathecal administration permits use ofan effective dose which is only a fraction of the effective doserequired by oral or parenteral administration. Furthermore, theintrathecal space is generally wide enough to accommodate a smallcatheter, thereby enabling chronic drug delivery systems. Thus, it isknown to treat spasticity by intrathecal administration of baclofen.Additionally, it is known to combine intrathecal administration ofbaclofen with intramuscular injections of botulinum toxin for theadjunct effect of intramuscular botulinum for reduced muscle spasticity.Furthermore, it is known to treat pain by intraspinal administration ofthe opioids morphine and fentanyl, as set forth in Gianno, J., et al.,Intrathecal Drug Therapy for Spasticity and Pain, Springer-Verlag(1996), the contents of which publication are incorporated herein byreference in its entirety.

[0011] The current method for intrathecal treatment of chronic pain isby use of an intrathecal pump, such as the SynchroMed® Infusion System,a programmable, implanted pump available from Medtronic, Inc., ofMinneapolis, Minn. A pump is required because the antinociceptive orantispasmodic drugs in current use have a short duration of activity andmust therefore be frequently readministered, which readministration isnot practically carried out by daily spinal tap injections. The pump issurgically placed under the skin of the patient's abdomen. One end of acatheter is connected to the pump, and the other end of the catheter isthreaded into a CSF filled subarachnoid or intrathecal space in thepatient's spinal cord. The implanted pump can be programmed forcontinuous or intermittent infusion of the drug through theintrathecally located catheter. Complications can arise due the requiredsurgical implantation procedure and the known intrathecally administereddrugs for pain have the disadvantages of short duration of activity,lipid solubility which permits passage out of the intrathecal space andsystemic transport and/or diffusion to higher CNS areas with potentialrespiratory depression resulting.

[0012] Thus, a significant problem with many if not all of the knownintrathecally administered drugs used to treat pain, whetheradministered by spinal tap or by catheterization, is that due to thedrug's solubility characteristics, the drug can leave the intrathecalspace and additionally due to poor neuronal binding characteristics, thedrug can circulate within the CSF to cranial areas of the CNS wherebrain functions can potentially be affected.

[0013] Botulinum Toxin

[0014] The anaerobic, gram positive bacterium Clostridium botulinumproduces a potent polypeptide neurotoxin, botulinum toxin, which causesa neuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack the central nervous system. The highest cranialnerves are affected first, followed by the lower cranial nerves and thenthe peripheral motor neurons. Symptoms of untreated botulinum toxinpoisoning can progress from and include medial rectus paresis, ptosis,sluggish pupillary response to light, difficulty walking, swallowing,and speaking, paralysis of the respiratory muscles and death.

[0015] Botulinum toxin type A is the most lethal natural biologicalagent known to man. It has been determined that 39 units per kilogram(U/kg) of intramuscular BOTOX®¹ is a LD₅₀ in primates. One unit (U) ofbotulinum toxin can be defined as the LD₅₀ upon intraperitonealinjection into mice. BOTOX® contains about 4.8 ng of botulinum toxintype A complex per 100 unit vial. Thus, for a 70 kg human a LD₅₀ ofabout 40 U/kg would be about 134 ng or 28 vials (2800 units) ofintramuscular BOTOX®. Seven immunologically distinct botulinumneurotoxins have been characterized, being respectively neurotoxinserotypes A, B, C1, D, E, F and G each of which is distinguished byneutralization with type-specific antibodies. The neurotoxin componentis noncovalently bound to nontoxic proteins to form high molecularweight complexes. The different serotypes of botulinum toxin vary in theanimal species that they affect and in the severity and duration of theparalysis they evoke. For example, it has been determined that botulinumtoxin type A is 500 times more potent, as measured by the rate ofparalysis produced in the rat, than is botulinum toxin type B.Additionally, botulinum toxin type B has been determined to be non-toxicin primates at a dose of 480 U/kg which is about 12 times the primateLD₅₀ for botulinum toxin type A (Moyer E et al., Botulinum Toxin Type B:Expenmental and Clinical Experience, being chapter 6, pages 71-85 of“Therapy With Botulinum Toxin”, edited by Jankovic, J. et al. (1994),Marcel Dekker, Inc.)

[0016] Minute quantities of botulinum toxin have been used to reduceexcess skeletal and smooth muscle and sphincter contraction. Thebotulinum toxin can be injected directly into the hyperactive orhypertonic muscle or its immediate vicinity and is believed to exert itseffect by entering peripheral, presynaptic nerve terminals at theneuromuscular junction and blocking the release of acetylcholine. Theaffected nerve terminals are thereby inhibited from stimulating musclecontraction, resulting in a reduction of muscle tone. Thus, wheninjected intramuscularly at therapeutic doses, botulinum toxin type Acan be used to produce a localized chemical denervation and hence alocalized weakening or paralysis and relief from excessive involuntarymuscle contractions.

[0017] Clinical effects of peripheral intramuscular botulinum toxin typeA are usually seen within one week of injection. The typical duration ofsymptomatic relief from a single intramuscular injection of botulinumtoxin type A averages about three months. Muscles therapeuticallytreated with a botulinum toxin eventually recover from the temporaryparalysis induced by the toxin, due possibly to the development of newnerve sprouts or to reoccurrence of neurotransmission from the originalsynapse, or both. A nerve sprout may establishes a new neuromuscularjunction. Thus, neuromuscular transmission can gradually return tonormal over a period of several months.

[0018] In skeletal and smooth muscle tissues botulinum toxin appears tohave no appreciable affinity for organs or tissues other thancholinergic neurons at the neuromuscular junction where the toxin bindsto and is internalized by neuronal receptors and, as indicated, blockpresynaptic release of the neurotransmitter acetylcholine, withoutcausing neuronal cell death.

[0019] Botulinum toxins have been used for the treatment of anincreasing array of disorders, relating to cholinergic nervous systemtransmission, characterized, for example, by hyperactive neuromuscularactivity in specific focal or segmental striated or smooth muscleregions. Thus, intramuscular injection of one or more of the botulinumtoxin serotypes has been used to treat, blepharospasm, spasmodictorticollis, hemifacial spasm, spasmodic dysphonia, oral mandibulardystonia and limb dystonias, myofacial pain, bruxism, achalasia,trembling chin, spasticity, juvenile cerebral palsy, hyperhydrosis,excess salivation, non-dystonic tremors, brow furrows, focal dystonias,tension headache, migraine headache and lower back pain. Notinfrequently, a significant amount of pain relief has also beenexperienced by such intramuscular therapy. These benefits have beenobserved after local intramuscular injection of, most commonly botulinumtoxin type A, or one or another of the other botulinum neurotoxinserotypes. Botulinum toxin serotypes B, C1, D, E and F apparently have alower potency and/or a shorter duration of activity as compared tobotulinum toxin type A at a similar dosage level.

[0020] Although all botulinum toxins serotypes apparently inhibitrelease of the neurotransmitter acetylcholine at the neuromuscularjunction, they do so by affecting different neurosecretory proteinsand/or cleaving these proteins at different sites. For example,botulinum types A and E both cleave the 25 kiloDalton (kD) synaptosomalassociated protein (SNAP-25), but they target different amino acidsequences within this protein. Botulinum toxin types B, D, F and G acton vesicle-associated protein (VAMP, also called synaptobrevin), witheach serotype cleaving the protein at a different site. Finally,botulinum toxin type C1 has been shown to cleave both syntaxin andSNAP-25. These differences in mechanism of action may affect therelative potency and/or duration of action of the various botulinumtoxin serotypes. The molecular weight of a secreted botulinum toxinprotein molecule, for all seven of the known botulinum toxin serotypes,is about 150 kD. Interestingly, the botulinum toxins are released byClostridial bacterium as complexes comprising the 150 kD botulinum toxinprotein molecule along with associated non-toxin proteins. Thus, thebotulinum toxin type A complex can be produced by Clostridial bacteriumas 900 kD, 500 kD and 300 kD forms. Botulinum toxin types B and C1 isapparently produced as only a 500 kD complex. Botulinum toxin type D isproduced as both 300 kD and 500 kD complexes. Finally, botulinum toxintypes E and F are produced as only approximately 300 kD complexes. Thecomplexes (i.e. molecular weight greater than about 150 kD) are believedto contain a non-toxin hemaglutinin protein and a non-toxin andnon-toxic nonhemaglutinin protein. These two non-toxin proteins (whichalong with the botulinum toxin molecule comprise the relevant neurotoxincomplex) may act to provide stability against denaturation to thebotulinum toxin molecule and protection against digestive acids whentoxin is ingested. Additionally, it is possible that the larger (greaterthan about 150 kD molecular weight) botulinum toxin complexes may resultin a slower rate of diffusion of the botulinum toxin away from a site ofintramuscular injection of a botulinum toxin complex.

[0021] The biochemical mechanism of the effects of botulinum toxin uponcentral nervous tissues is controversial. Additionally, the number ofCNS neurotransmitters affected as well as the extent and nature of theeffect of botulinum toxin upon the synthesis, release, accumulation andmetabolism of different CNS neurotransmitters is still being determined.In vitro studies have indicated that botulinum toxin inhibits potassiumcation induced release of both acetylcholine and norepinephrine fromprimary cell cultures of brain tissue. Additionally, it has beenreported that botulinum toxin inhibits the evoked release of bothglycine and glutamate in primary cultures of spinal cord neurons andthat in brain synaptosome preparations botulinum toxin inhibits therelease of each of the neurotransmitters acetylcholine, dopamine,norepinephrine, CGRP and glutamate.

[0022] Botulinum toxin type A can be obtained by establishing andgrowing cultures of Clostridium botulinum in a fermenter and thenharvesting and purifying the fermented mixture in accordance with knownprocedures. All the botulinum toxin serotypes are initially synthesizedas inactive single chain proteins which must be cleaved or nicked byproteases to become neuroactive. The bacterial strains that makebotulinum toxin serotypes A and G possess endogenous proteases andserotypes A and G can therefore be recovered from bacterial cultures inpredominantly their active form. In contrast, botulinum toxin serotypesC1, D and E are synthesized by nonproteolytic strains and are thereforetypically unactivated when recovered from culture. Serotypes B and F areproduced by both proteolytic and nonproteolytic strains and thereforecan be recovered in either the active or inactive form. However, eventhe proteolytic strains that produce, for example, the botulinum toxintype B serotype only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin is likely to be inactive, possibly accounting for the knownsignificantly lower potency of botulinum toxin type B as compared tobotulinum toxin type A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that botulinum toxin type B has, uponintramuscular injection, a shorter duration of activity and is also lesspotent than botulinum toxin type A at the same dose level.

[0023] What is needed therefore is a method for effectively treatingpain and/or spasm by intraspinal administration of a pharmaceuticalwhich has the characteristics of long duration of activity, low rates ofdiffusion out of an intrathecal space where administered, low rates ofdiffusion to other intrathecal areas outside of the site ofadministration, specificity for the treatment of pain and limited orinsignificant side effects at therapeutic dose levels.

SUMMARY

[0024] The present invention meets this need and provides methods foreffectively treating pain by intraspinal administration of a neurotoxinwhich has the characteristics of long duration of activity, low rates ofdiffusion out of an, for example, intrathecal space where administered,low rates of diffusion to other intrathecal areas outside of the site ofadministration, specificity for the treatment of pain and limited orinsignificant side effects at therapeutic dose levels. A method fortreating pain according to the present invention can have the step ofintraspinal administration of a neurotoxin to a mammal, therebyalleviating pain experienced by the mammal. Preferably, the neurotoxinused is a botulinum toxin, such as one of, or a combination of one ormore, of the botulinum toxin serotypes A, B, C, D, E, F and G. Mostpreferably, the botulinum toxin used is botulinum toxin type A becauseof the high potency, ready availability and long history of clinical useof botulinum toxin type A to treat various disorders.

[0025] The neurotoxin intraspinally administered according to themethods of the present invention has not been conjugated, attached,adhered to or fused to and is not administered in conjunction with aneuronal targeting moiety. A neuronal targeting moiety is a compoundwhich functionally interacts with a binding site on a neuron causing aphysical association between the targeting moiety and/or a conjugateattached to the targeting moiety and the surface of the neuron, such asa primary sensory afferent. Thus, the targeting moiety providesspecificity for or binding affinity for one or more type of neurons. Inthe present invention, any pharmaceutical preparation (i.e. areconstituted solution of neurotoxin, sodium chloride (saline) and astabilizer such as albumin) which incorporates a neurotoxin for useaccording to the disclosed methods is devoid of or essentially free ofany deliberately attached or prepared neuronal targeting moiety.

[0026] Use of one or more targeting moiety artifacts or constructs isexcluded from the scope of the present invention as unnecessary becausewe have surprisingly discovered that intraspinal neurotoxinadministration according to the present invention provides significantpain alleviation even though the neurotoxin is not administrated inconjunction with any non-native or non-inherent to the neurotoxinneuronal targeting moiety. Thus, we unexpectedly discovered that anative neurotoxin, such as botulinum toxin type A, can upon intraspinaladministration interact with neurons of the CNS and provide alleviationof pain even though the neurotoxin has not been artificially ormanipulatively accorded any neuronal specificity or binding affinity,such as by attachment of a neuronal targeting moiety to the neurotoxin.Prior to our invention, it has been believed, as discussed infra, that aneurotoxin, such as botulinum toxin type A, would upon intraspinal,including intrathecal, administration, exert widespread, unfocused,diffuse and deleterious effects upon the CNS, such deleterious effectsincluding spasticity. Hence, the assumed necessity for a neuronaltargeting moiety deliberately attached to the neurotoxin to attenuate oreliminate these presumed detrimental effects resulting from intraspinaladministration of a neurotoxin, such a botulinum toxin type A.

[0027] We have surprising found that a botulinum toxin, such asbotulinum toxin type A, can be intraspinally administered in amountsbetween about 10⁻³ U/kg and about 60 U/kg to alleviate pain experiencedby a mammal, such as a human patient. Preferably, the botulinum toxinused is intraspinally administered in an amount of between about 10⁻²U/kg and about 50 U/kg. More preferably, the botulinum toxin isadministered in an amount of between about 10⁻¹ U/kg and about 40 U/kg.Most preferably, the botulinum toxin is administered in an amount ofbetween about 1 U/kg and about 30 U/kg. In a particularly preferredembodiment of the present disclosed methods, the botulinum toxin isadministered in an amount of between about 1 U/kg and about 20 U/kg andin some clinical settings the botulinum toxin can advantageously beadministered in an amount of between about 1 U/kg and about 10 U/kg.Significantly, the pain alleviating effect of the present disclosedmethods can persist for up to 10 days or for up to 20 days and dependingupon factors, such as the dosage used, for up to 3 months per neurotoxinadministration.

[0028] The intraspinal administration of the neurotoxin is preferably byintrathecal administration, such as intrathecally to a cranial,cervical, thoracic, lumbar, sacral or coccygeal region of the centralnervous system and the administration step can include the steps ofaccessing a subarachnoid space of the central nervous system of themammal, and injecting the neurotoxin into the subarachnoid space. Theaccessing step can be carried out by effecting a spinal tap.

[0029] Alternately, the intraspinal administration step can include thesteps of catheterization of a subarachnoid space of the central nervoussystem of the mammal, followed by injection of the neurotoxin through acatheter inserted by the catheterization step into the subarachnoidspace. Note that prior to the injecting step there can be the step ofattaching to or implanting in the mammal an administration means foradministering the neurotoxin to the central nervous system of themammal. The administration means can be made up of a reservoir of theneurotoxin, where the reservoir is operably connected to a pump meansfor pumping an aliquot of the neurotoxin out of the reservoir and intoan end of the catheter in the subarachnoid space.

[0030] It is important to note that the administration step can becarried out prior to the onset of or subsequent to the occurrence of anociceptive (inflammatory, neuropathic, injury induced, resulting form acancer, spasm, etc) event or syndrome experienced by the mammal. Thus,the administration step can be carried out between about more than 0.5hour before to about 14 days before the onset of the nociceptive event.More preferably, administration step is carried out between about morethan 0.5 hour before to about 10 days before the onset of thenociceptive event. Most preferably, the administration step is carriedout between about more than 0.5 hour before to about 7 days, 4 days, 24hours or 6 hours before the onset of the nociceptive event. In aparticularly preferred embodiment of the present invention, theadministration step is carried out between about 2 hours before to about5 hours before the onset of the nociceptive event. The present methodscan be used to treat the pain associated with allodynia.

[0031] A detailed embodiment of a method within the scope of the presentinvention can include the steps of firstly catheterization of asubarachnoid space of the central nervous system of the mammal by makingan incision though the dermis of the mammal, and then threading acatheter through the incision into the subarachnoid space, the catheterhaving an open first end and a remote open second end. Secondly,attaching to or implanting in the mammal an administration means foradministering a botulinum toxin to the subarachnoid space of the centralnervous system of the mammal, the administration means comprising areservoir for holding a multidose amount of the botulinum toxin, thereservoir being connected to a pump means for pumping an aliquot of thebotulinum toxin out of the reservoir and into the first end of acatheter, the first end of the catheter being connected to the pumpmeans. Thirdly, activating the pump means, and finally, injecting intothe subarachnoid space of the central nervous system of the mammal andthrough the second end of the catheter between about 10⁻¹ U/kg and about60 U/kg of the botulinum toxin, thereby alleviating pain experienced bythe mammal.

[0032] Another preferred method within the scope of the presentinvention is a method for the in vivo attenuation of a nociceptiveactivity or experience of a human patient, the method comprising thestep of intraspinal administration to a human patient a therapeuticallyeffective amount of a botulinum toxin, thereby causing an in vivoattenuation of a nociceptive activity or experience of the humanpatient. The intraspinal administration step can be carried outsubsequent to or prior to the occurrence or onset of a nociceptiveactivity, experience, sensation or syndrome.

[0033] A further preferred method within the scope of the presentinvention is a method for treating pain by selecting a neurotoxin withantinociceptive activity, choosing a portion of a central nervous systemof a patient which influences a nociceptive activity; and intraspinallyadministering to the portion of the central nervous system chosen theneurotoxin selected.

[0034] Notably, the neurotoxin used to practice the present methods canbe made by a Clostridial bacterium, such as one or more of theClostridium botulinum, Clostridium butyricum, and Clostridium berattispecies.

[0035] Another preferred method within the scope of the presentinvention is a method for treating pain, the method comprising the stepof administering a neurotoxin to the central nervous system or to adorsal root ganglion of a mammal, thereby alleviating pain experiencedby the mammal. A further preferred method within the scope of thepresent invention is a method for improving patient function, the methodcomprising the step of administering a neurotoxin to the central nervoussystem or to dorsal root ganglion of a mammal, thereby improving patientfunction as determined by improvement in one or more of the factors ofreduced pain, reduced time spent in bed, increased ambulation, healthierattitude and a more varied lifestyle.

[0036] The present invention also includes within its scope a methodwhich uses a modified neurotoxin. By a modified neurotoxin it is meant aneurotoxin which has had one or more of its amino acids deleted,modified or replaced (as compared to the native neurotoxin) and includesrecombinant technology made neurotoxins as well as derivatives andfragments of a recombinant produced neurotoxin.

DRAWINGS

[0037] These and other features, aspects, and advantages of the presentinvention can become better understood from the following description,claims and the accompanying drawings, where in all of FIGS. 1-7 below,“injection” means intrathecal injection.

[0038]FIG. 1 is a dose response graph showing that a method within thescope of the present invention alleviates induced inflammatory painunder the rat formalin model. The x axis set forth time in minutes aftercommencement of the formalin model in rats. The y axis sets forth timespent lifting and licking the formalin injected paw upon use of control(saline, n=11) and BOTOX® (botulinum toxin Type A purified neurotoxincomplex) injections at concentrations of 0.0625 U/kg (n=10), 0.625 U/kg(n=14) and 3.125 U/kg (n=9) injected from 2 hours to 5 hours beforecommencement of the formalin challenge.

[0039]FIG. 2 is a time course graph showing that a method within thescope of the present invention alleviates induced inflammatory painunder the rat formalin model for at least seven days when injected morethan one half hour before commencement of the formalin test. The x axisset forth time in minutes after commencement of the formalin model inrats. The y axis sets forth time spent lifting and licking the formalininjected paw upon use of control (saline, n=8) and BOTOX® injections ata concentration of 0.625 U/kg injected 0.5 hour before, 2 hours to 5hours before (n=14) and 7 days before (n=5) commencement of the formalinchallenge.

[0040]FIG. 3 is a dose response graph showing that a method within thescope of the present invention alleviates induced inflammatory painunder the rat formalin model for at least seven days when differentconcentrations of botulinum toxin type A are used. The x axis set forthtime in minutes after commencement of the formalin model in rats. The yaxis sets forth time spent lifting and licking the formalin injected pawupon use of control (saline, n=11) and BOTOX® injections atconcentrations of 0.0625 U/kg injected 7 days before (n=8), 0.625 U/kginjected 7 days before (n=7) and 3.125 U/kg injected 7 days before (n=6)commencement of the formalin challenge.

[0041]FIG. 4 is a time course graph showing that a method within thescope of the present invention alleviates induced inflammatory pain inthe rat formalin model. The x axis set forth time in minutes aftercommencement of the formalin model in rats. The y axis sets forth timespent lifting and licking the formalin injected paw upon injection ofcontrol (saline, n=11), and BOTOX® at a concentration of 0.625 U/kginjected 2 hours 14 days before (n=4) commencement of the formalinchallenge.

[0042]FIG. 5 is a graph which shows a comparison of the analgesic effectof botulinum toxin type A and muscimol upon induced inflammatory pain inthe rat formalin model. The x axis set forth time in minutes aftercommencement of the formalin model in rats. The y axis sets forth timespent lifting and licking the formalin injected paw upon use of controlinjection (saline, n=11), BOTOX® at a concentration of 0.625 U/kginjected 2 hours to 5 hours before or six days before, and 1 μg ofmuscimol injected 10 minutes before or six days before commencement ofthe formalin challenge.

[0043]FIG. 6 is graph showing that a method within the scope of thepresent invention alleviates induced inflammatory pain in the ratformalin model with a local intrathecal analgesic effect. The x axis setforth time in minutes after commencement of the formalin model in rats.The y axis sets forth time spent lifting and licking the formalininjected paw upon use of control where the catheter used forintrathecally injecting saline was located either 4.5 cm (n=1) or 8.5 cm(n=11) caudally (at the lumbar enlargement therefore) from its insertionpoint, and BOTOX® at a concentration of either 0.625 U/kg (n=3) or 3.125U/kg (n=4) was injected through a catheter located only 4.5 cm caudallyfrom its insertion point.

[0044]FIG. 7 is a graph showing that a method within the scope of thepresent invention alleviates surgically induced neuropathic pain. The xaxis sets forth time in hours after injection of either saline (n=8) orBOTOX® at a concentration of 0.625 U/kg (n=11) or 3.125 U/kg (n=9). They axis sets forth the G value, a measure of analgesic effect. BL meansbaseline.

DESCRIPTION

[0045] The present invention encompasses methods for treating pain. Wehave discovered that intraspinal administration of a neurotoxin to thecentral nervous system of a patient can result in significant and longlasting alleviation of pain without significant undesirable sideeffects. Thus, a method within the scope of the present inventionprovides antinociceptive or analgesic relief.

[0046] As used herein “intraspinal” means into or within the epiduralspace, the intrathecal space, the white or gray matter of the spinalcord or affiliated structures such as the dorsal root and dorsal rootganglia.

[0047] Prior to our invention it had been believed by those skilled inthe art that intrathecal administration of a neurotoxin, such as abotulinum toxin, would (1) induce significant spasticity in therecipient and (2) promote detrimental effects upon spinal cord and brainfunctions. Thus, with regard to cited deleterious effect (1): it wasreported, as examples, in Williamson et al., in Clostridial Neurotoxinsand Substrate Proteolysis in Intact Neurons, J. of Biological Chemistry271:13; 7694-7699 (1996) that both tetanus toxin and botulinum toxintype A inhibit the evoked release of the neurotransmitters glycine andglutamate from fetal mice spinal cord cell cultures, while it wasreported by Hagenah et al., in Effects of Type A Botulinum Toxin on theCholinergic Transmission at Spinal Renshaw Cells and on the InhibitoryAction at la Inhibitory Intemeurones, Naunyn-Schmiedeberg's Arch.Pharmacol. 299, 267-272 (1977), that direct intraspinal injection ofbotulinum toxin type A in experimentally prepared, anaesthetized catsinhibits CNS Renshaw cell activity. Inhibition of central glycine andglutamate neurotransmitter release as well as the downregulation ofRenshaw cell activity presumably can both result in vivo in thepromotion of significant motorneuron hyperactivity with ensuingperipheral muscle spasticity.

[0048] With regard to deleterious effect (2): it is believed thatintrathecal administration of the tetanus neurotoxin exerts, byretrograde movement of the tetanus toxin along CNS neurons, significantnegative effects upon spinal cord and brain functions, therebycontraindicating intrathecal administration of a related neurotoxin,such as a botulinum toxin. Notably, botulinum toxin and tetanus toxinare both made by Clostridial bacteria, although by different species ofClostridium. Significantly some researchers have reported that botulinumtoxin shares, at least to some extent, the noted neural ascentcharacteristic of tetanus toxin. See e.g. Habermann E., ¹²⁵ I-LabeledNeurotoxin from Clostridium Botulinum A: Preparation, Binding toSynaptosomes and Ascent in the Spinal Cord, Naunyn-Schmiedeberg's Arch.Pharmacol. 281, 47-56 (1974).

[0049] Our invention surprisingly encounters neither of the deleteriouseffects (1) or (2), and the disclosed methods of the present inventioncan be practiced to provide effective and long lasting relief from painand to provide a general improvement in the quality of life experiencedby the treated patient. The pain experienced by the patient can be due,for example, to injury, surgery, infection, accident or disease(including cancer and diabetes), including neuropathic diseases anddisorders.

[0050] Preferably, a neurotoxin used to practice a method within thescope of the present invention is a botulinum toxin, such as one of theserotype A, B, C, D, E, F or G botulinum toxins. Preferably, thebotulinum toxin used is botulinum toxin type A, because of its highpotency in humans, ready availability, and known use for the treatmentof skeletal and smooth muscle disorders when locally administered byintramuscular injection. Botulinum toxin type B is not a preferred toxinto use in the practice of the disclosed methods because type B is knownto have a significantly lower potency and efficacy as compared, to typeA, is not readily available, and has a limited history of clinical usein humans.

[0051] An intraspinal route for administration of a neurotoxin accordingto the present disclosed invention can be selected based upon criteriasuch as the solubility characteristics of the neurotoxin toxin chosen aswell as the amount of the neurotoxin to be administered. The amount ofthe neurotoxin administered can vary widely according to the particulardisorder being treated, its severity and other various patient variablesincluding size, weight, age, and responsiveness to therapy. For example,the extent of the area of CNS afferent pain neuron somata influenced isbelieved to be proportional to the volume of neurotoxin injected, whilethe quantity of the analgesia is, for most dose ranges, believed to beproportional to the concentration of neurotoxin injected. Furthermore,the particular intraspinal location for neurotoxin administration candepend upon the dermosome location of the pain to be treated. Methodsfor determining the appropriate route of administration and dosage aregenerally determined on a case by case basis by the attending physician.Such determinations are routine to one of ordinary skill in the art (seefor example, Harrison's Principles of Internal Medicine (1997), editedby Anthony Fauci et al., 14^(th) edition, published by McGraw Hill).

[0052] Preferably, the intraspinal administration is carried outintrathecally because of the greater ease in which the relatively largerintrathecal space is accessed and because the preferred neurotoxin, abotulinum toxin, generally exhibits low solubility in the lipid richepidural environment. Additionally, epidural neurotoxin administrationis a less preferred route of intraspinal administration because theneurotoxin must diffuse through the intrathecal space to have anantinociceptive effect by, it is believed, action upon neurons of theCNS and dorsal root ganglia (DRG). We have found that both inflammatoryand neuropathic pain can be effectively treated by the disclosed methodswithout significant muscle spasticity or flaccidity or other sideeffects.

[0053] Intraspinal administration of a neurotoxin according to thepresent invention can be by various routes such as by catheterization orby spinal tap injection. The long lasting nature of the therapeuticeffects of the present invention substantially removes the need forchronic antinociceptive drug administration, so that the present methodsare advantageously practiced by infrequent spinal tap injection of theneurotoxin. Additionally, an intrathecal spinal tap neurotoxinadministration route facilitates a more precise and localized deliveryof toxin with less danger of damage to the CNS, as compared to moving acatheter to access other CNS locations.

[0054] Intrathecal neurotoxin can be administered by bolus injection orby catheterization. The catheter can be inserted at L3-4 or at L4-5, asafe distance from the spinal cord which in humans terminates at L1, andguided upward in the subarachnoid space to rest at the desired site. Forpain management, placement of the catheter or location of bolusinjection by syringe depends on the site of the perceived pain, and thephysicians preference.

[0055] It is important to note that therapeutic neurotoxinadministration according to the present disclosed methods can be carriedout before the occurrence of or during the experience of a nociceptiveevent or syndrome.

[0056] We have found that a neurotoxin, such as a botulinum toxin, canbe intraspinally administered according to the present disclosed methodsin amounts of between about 10⁻³ U/kg to about 60 U/kg. A dose of about10⁻³ U/kg can result in an antinociceptive effect if delivered directlyto or onto the dorsal horn of the CNS and/or if botulinum toxin deliveryis assisted by methods such as iontophoresis. Intraspinal administrationof less than about 10⁻³ U/kg does not result in a significant or lastingtherapeutic result. An intraspinal dose of more than 60 U/kg approachesa lethal dose of a neurotoxin such as a botulinum toxin. It is desiredthat the neurotoxin used to obtain either antinociceptive effect contactthe nerves of the CNS so as to favorably influence or down regulate theperception of pain or muscle spasm in the innervated organ or tissue.Thus, intraspinal administration of a neurotoxin by, for example,epidural injection can require an increase of the dosage by a factor ofabout ten to account for dilution of the neurotoxin upon diffusion fromthe epidural space to the intrathecal space and thence to the exteriornerves of the CNS.

[0057] A preferred range for intrathecal administration of a botulinumtoxin, such as botulinum toxin type A, so as to achieve anantinociceptive effect in the patient treated is from about 10⁻² U/kg toabout 50 U/kg. Less than about 10⁻² U/kg result in a relatively minor,though still observable, antinociceptive effects, while more than about50 U/kg can result in some muscle flaccidity and symptoms of toxinintoxication. A more preferred range for intrathecal administration of abotulinum toxin, such as botulinum toxin type A, so as to achieve anantinociceptive effect in the patient treated is from about 10⁻¹ U/kg toabout 30 U/kg. Less than about 10⁻¹ U/kg can result in the desiredtherapeutic effect being of less than the optimal or longest possibleduration, while more than about 30 U/kg can still result in somesymptoms of muscle flaccidity. A most preferred range for intrathecaladministration of a botulinum toxin, such as botulinum toxin type A, soas to achieve an antinociceptive effect in the patient treated is fromabout 1 U/kg to about 20 U/kg. Intrathecal administration of a botulinumtoxin, such as botulinum toxin type A, in this preferred range canprovide dramatic therapeutic success. Furthermore, our experimental workindicates that a dose range of about 1 U/kg to about 10 U/kg can providesignificant and long lasting antinociceptive effect without significantside effects for the treatment of inflammatory and neuropathic pain inhuman patients.

[0058] We have determined by immunohistochemical staining of cleavedSNAP-25 proteins produced by BOTOX®, that intrathecally administeredBOTOX® distributes in the superficial layer of the rat dorsal horn,which is the spinal cord layer in which afferent pain fibers terminate.Thus, without wishing to be bound to any particular theory, wehypothesize that the antinociceptive effect of intrathecal botulinumtoxin is due to its specific inhibition of the release of variousneurotransmitters from central terminal afferent sensory neurons and/orfrom second order projecting neurons in the dorsal horn. The presentinvention includes within its scope the use of any neurotoxin which hasa long duration antinociceptive effect when locally applied to thecentral nervous system of a patient. For example, neurotoxins made byany of the species of the toxin producing Clostridium bacteria, such asClostridium botulinum, Clostridium butyricum, and Clostridium berattican be used or adapted for use in the methods of the present invention.Additionally, all of the botulinum serotypes A, B, C, D, E, F and G canbe advantageously used in the practice of the present invention,although type A is the most preferred and type B the least preferredserotype, as explained above. Practice of the present invention canprovide an analgesic effect, per injection, for 3 months or longer inhumans.

[0059] Significantly, a method within the scope of the present inventioncan provide improved patient function. “Improved patient function” canbe defined as an improvement measured by factors such as a reduced pain,reduced time spent in bed, increased ambulation, healthier attitude,more varied lifestyle and/or healing permitted by normal muscle tone.

[0060] As set forth above, we have discovered that a surprisinglyeffective and long lasting treatment of pain can be achieved byintraspinal administration of a neurotoxin to an afflicted patient. Inits most preferred embodiment, the present invention is practiced byintrathecal injection of botulinum toxin type A. Significantly, we havediscovered that dramatic, long term analgesic and/or improved patientfunction effects can be achieved through intraspinal administration of aneurotoxin by the methods disclosed herein even though the neurotoxinhas not had attached or fused to it, by various manipulative techniquesor technologies, a neuronal targeting moiety, such as a non-neurotoxinprotein, to provide targeting specificity of the neurotoxin for one ormore particular types of neurons. Thus, the present invention excludesfrom its scope the use of any neurotoxins with one or more artificiallyattached or fused neuronal targeting moieties. A neurotoxin can displaya natural binding affinity for a neuron (i.e. for a particular receptoron the surface of the neuron) due to the presence of a binding moietyinherent to the structure of the native neurotoxin molecule (forexample, the binding domain of the heavy chain of a botulinum toxin,i.e. the H_(C) fragment). Thus, for clarity “targeting moiety” or“neuronal targeting moiety” as used herein means a targeting moietywhich provides to a neurotoxin specific or enhanced neuronal bindingaffinity and which is not a natural or inherent feature of theneurotoxin which has such a targeting moiety. Contrarily, “bindingmoiety” as used herein means the inherent component or domain of thenative neurotoxin which provides neuronal binding affinity.

[0061] The present invention does include within its scope: (a)neurotoxin obtained or processed by bacterial culturing, toxinextraction, concentration, preservation, freeze drying and/orreconstitution and; (b) modified or recombinant neurotoxin, that isneurotoxin that has had one or more amino acids or amino acid sequencesdeliberately deleted, modified or replaced by known chemical/biochemicalamino acid modification procedures or by use of known hostcell/recombinant vector recombinant technologies, as well as derivativesor fragments of neurotoxins so made, but, as stated, excludesneurotoxins with one or more attached neuronal targeting moieties.

[0062] Botulinum toxins for use according to the present invention canbe stored in lyophilized or vacuum dried form in containers under vacuumpressure. Prior to lyophilization the botulinum toxin can be combinedwith pharmaceutically acceptable excipients, stabilizers and/orcarriers, such as albumin. The lyophilized material can be reconstitutedwith saline or water.

EXAMPLES

[0063] The following examples provide those of ordinary skill in the artwith specific preferred methods within the scope of the presentinvention for carrying out the present invention and are not intended tolimit the scope of what the inventors regards as their invention.Examples 1-4 and 6 show that intrathecal administration of botulinum Ahas an analgesic effect upon inflammatory pain while examples 5 and 7show that intrathecal administration of botulinum A has an analgesiceffect upon neuropathic pain.

Example 1 Analgesic Effect of Intrathecally Administered Botulinum ToxinType A Upon Inflammatory Pain

[0064] The purpose of this experiment was to investigate the analgesiceffect of botulinum toxin type A on inflammatory pain using the ratformalin model.

[0065] Male Sprague-Dawley rats weighing 270 g to 350 g each wereanesthetized with isoflurane. In this and in all subsequent Examplesintrathecal administration of a neurotoxin was carried out byintrathecal cannulation performed by inserting a PE (polyethylene)-10tubing about 10 cm long through an incision in the dura over thecisterna and threaded caudally about 8.5 cm down the spinal cord of therat to the vicinity of the lumbar enlargement, as described in Yaksh T.et al., Chronic Catheterization of the Spinal Subarachnoid Space, Physio& Behav 17: 1031-1036 (1976). Either BOTOX® or the control fluid salinewas administered intrathecally through the lumbar enlargement locatedcatheter from 0 to 5 hours before the formalin test.

[0066] The formalin test to assess analgesia, as set forth in DubuissonD., et al The Fonmalin Test: A Quantitative Study of the AnalgesicEffects of Morphine, Merperdine, and Brain Stem Stimulation in Rats andCats, Pain, 4 (1977), 161-174, was followed. Thus, formalin (5%, 50 μl)was injected subcutaneously into rat's right hind paw. We evaluated thenumber of formalin-evoked flinching responses and the time spent lickingthe injected paw during time intervals. In the formalin test, recordingof the early response (early phase) starts immediately and lasted for 5min (0-5 min). The recording of the late response (late phase) starts 10min after formalin injection and lasts for 50 min (10-60 min).

[0067]FIG. 1 shows that intrathecal administration of BOTOX® (0.0625U/kg, 0.625 U/kg or 3.125 U/kg) 2-5 hrs before injection of the formalinreduced the inflammatory pain induced by the formalin model. The controlgroup (n-11) was treated with saline intrathecally. Injection offormalin in rat right hind paw produced a consistent lift/licking andflinch response in the both first 5 min (first phase) and 10-60 min(second phase). BOTOX®) at doses of 0.0625 U/kg (0.003 ng/kg; n=10),0.625 U/kg (0.03 ng/kg, n=14) and at 3.125 U/kg (0.15 ng/kg, n=9)significantly decreased the lift/licking time during first and secondphase. By convention one unit (U) of reconstituted BOTOX® provides amedian lethal intraperitoneal dose (LD₅₀) in mice.

[0068] The first phase (from time 0 to about plus 5-10 minutes inFIG. 1) is believed to be representative of a short lasting burst ofunmyelinated primary afferent neuron activity. In the longer secondphase (from about time plus 5-10 minutes in FIG. 1), it is believed thatan extended low level of C-fiber activity produces a facilitation inwhich the output of the WDR (DR meaning dorsal root) neuron is muchexaggerated relative to the C-fiber input.

[0069] This example shows that intrathecal administration of botulinumtoxin type had a significant analgesic effect on inflammatory pain atdoses of 0.0625 U/kg (0.003 ng/kg; n=10), 0.625 U/kg (0.03 ng/kg, n=14)and 3.125 U/kg (0.15 ng/kg, n=9) as measured by significantly decreasedthe lift/licking time during first and second phases.

Example 2 Analgesic Effect of Intrathecally Administered Botulinum ToxinType A Upon Inflammatory Pain Persists For At Least Fourteen Days

[0070] Intrathecal cannulation of male Sprague-Dawley rats was carriedout as set forth in Example 1. FIG. 2 (control, n=8) shows thatpretreatment of rats with BOTOX® (0.03 ng/kg or 0.625 U/kg, n=14) 2 to 5hrs before injection of formalin reduced the lift/licking time in boththe first and second phases. The analgesic effect of BOTOX® persistedfor 7 days (0.625 U/kg, n=5) after treatment with BOTOX®, but isdiminished compared to the 2 hr pre-treatment (FIG. 2). Additionally, asshown by FIG. 3, the analgesic effect at day 7 after intrathecalbotulinum type A administration is dose dependant. Furthermore, as shownby FIG. 4, the analgesic effect of BOTOX® persists for at least 14 days(0.625 u/KG, N=4). As shown by FIG. 2, pretreatment of rats with BOTOX®0.5 hr before initiation of the formalin challenge failed to reduce theformalin-induced pain.

[0071] This example shows that a significant analgesic effect ofintrathecal botulinum toxin type A persist for at least 14 days in ratsafter administration of the toxin. It can be reasonably postulated,extrapolating from the data obtained, that the analgesia persists for atleast about 20 days in rats. It can therefore by expected that ananti-inflammatory pain analgesia from intrathecal administration ofbotulinum toxin type A in humans would persist for at least about 60days.

Example 3 Comparison of Analgesic Effects of Intrathecally AdministeredBotulinum Toxin Type A and Muscimol Upon Inflammatory Pain.

[0072] Intrathecal cannulation of subject rats was carried out as setforth in Example 1. Either BOTOX® (0.625 U/kg, 2-5 hours before or sixdays before the formalin test) or the short acting analgesic muscimol (1μg, 10 minutes before or six days before the formalin test) wasadministered intrathecally and the formalin test carried out at theindicated subsequent times.

[0073] As shown by FIG. 5 (control saline, n=11), the analgesic effectof BOTOX® administered six days prior to the formalin test has a longerduration of analgesic activity, through most of phase 2, as compared tothe analgesic effect of intrathecal muscimol administered six days priorto the formalin test. Additionally, FIG. 5 shows that intrathecal BOTOX®administered 2-5 hours before the formalin challenge and intrathecalmuscimol ten minutes prior to the formalin challenge resulted incomparable analgesia.

Example 4 Site Specific Analgesic Effect of Intrathecally AdministeredBotulinum Type A Upon Inflammatory Pain

[0074] Intrathecal canalization was carried out as set forth in Example1 with the exception that the catheter was inserted caudally only about4.5 cm, as opposed to the usual 8.5-10 cm. Control (saline) catheterswere inserted at either 8.5 cm (n=11) or at 4.5 cm (n=1) locations.BOTOX® was administered through a catheter inserted caudally 4.5 cm indosages of either 0.625 U/kg (n=3) or 3.125 U/kg (n=4). The rat formalintest was then carried out. As shown by FIG. 6, there was little or noanalgesic effect in the rat formalin test by intrathecal BOTOX®administration through catheters placed at 4.5 cm.

[0075] It is known that the heel and bottom of the foot in humans is adermatome of the fifth lumbar nerve which emanates from the lumbarenlargement (see e.g. plate 150 in Netter, F. Atlas of Human Anatomy,second edition (1997), Novartis), and presumably nerve distribution issimilar in the rat. Thus, it can be hypothesized that since the ratplantar, where the formalin is injected, is innervated by nerves whichradiate from the lumbar enlargement disposed about 7.5 cm to 9 cm(depending upon the size of the subject rat) caudally down the rat'sspinal cord, placement of the intrathecal catheter caudally only 4.5 cmwill not result in an analgesic effect if the intrathecally administeredBOTOX® exhibits a site specific effect upon spinal cord neurons. Andthis hypothesis is confirmed by the data shown in FIG. 5.

[0076] This example supports both the efficacy and safety of intrathecalbotulinum toxin administration to treat pain since we observed that notonly did neither a motor deficit or blood pressure alteration occur, atthe dosages used, from intrathecal BOTOX® administration, we alsodetermined (FIG. 5) that intrathecal BOTOX® apparently has a localizedeffect upon the CNS at only the site of it's intrathecal administration.

Example 5 Analgesic Effect of Intrathecally Administered Botulinum ToxinType A Upon Neuropathic Pain

[0077] This example investigated whether botulinum toxin type A couldreduce the allodynia induced by L5, L6 nerve ligation. Male SpragueDawley rats (100-120 g) were anesthetized with isoflurane following asurgical neuropathy procedure according to the method set forth in KimS. et al., An Experimental Model for Peripheral Neuropathy Produced bySegmental Spinal Nerve Ligation in the Rat, Pain, 50 (1992), 355-363.The L6 transverse process was exposed and removed. The L4 and L5 spinalnerves were then isolated and visible and the ligation of L5 wasperformed by tying tightly with a 3-0 silk thread. The L6 spinal nervewas located just caudal and medial to the sacroiliac junction and wasligated with 6-0 suture. Intrathecal cannulation (as set forth inExample 1) was carried out a month later upon the rats which exhibitedallodynia.

[0078] Deformities of the hind paw and growth of the toenails werenoticed after surgery. Rats developed allodynia by showing sensitiveresponse to normally innocuous mechanical stimuli using the followingprotocol. Tactile allodynia was measured using von Frey hairaesthesiometers. The rats were tested before (Baseline) and afteradministration of the botulinum toxin type A as BOTOX®. Testing wasperformed during only the day portion of the circadian cycle. Rats wereplaced in a plastic cage with a wire mesh bottom which allowed fullaccess to the paws. Environmental acclimation was allowed forapproximately 30 minutes until cage exploration and major groomingactivities ceased. The area tested was the mid plantar left hind paw inthe sciatic nerve distribution, avoiding the less sensitive tori (footpads). The paw was touched with one of a series of 8 von Frey hairs withexperimentally incremental stiffness (0.41, 0.70, 1.20, 2.00, 3.63,5.50, 8.50, and 15.10 g) (Stoelting). The von Frey hair was presentedperpendicular to the plantar surface with sufficient force to causeslight buckling against the paw and held for approximately 6-8 seconds.Stimuli were presented at intervals of several seconds allowing forapparent resolution of any behavioral responses to previous stimuli. Apositive response was noted if the paw was sharply withdrawn. Ambulatingwas considered an ambiguous response, and in such cases the stimulus wasrepeated. Based on observations on normal, unoperated on rats andhealed, sham-operated rats, the cutoff of a 15.10 g hair (approximately10% of the body weight of the smaller rats) was selected as the upperlimit for testing, since stiffer hairs tended to raise the entire limbrather than to buckle, thus substantially changing the nature of thestimulus.

[0079] The 50% withdraw threshold (G Value) was determined using theup-down method (Dixon W., Efficient Analysis of ExperimentalObservations, Ann Rev Pharmacol Toxicol 1980, 20: 441-62). In thisparadigm testing is initiated with the 2.0 g hair, the middle hair ofthe series. Stimuli are always presented in a consecutive fashion,whether ascending or descending. In the absence of a paw withdrawalresponse to the initially selected hair a stronger stimulus ispresented. If the paw is withdrawn then the next weaker stimulus ischosen. Optimal threshold calculation by this method requires sixresponses in the immediate vicinity of the 50% threshold. Since thethreshold is not known strings of similar responses may be generated asthe threshold is approached from either direction. Accordingly, althoughall responses are noted, counting of the critical six data points doesnot begin until the response threshold has been crossed, at which timethe two responses straddling the threshold are retrospectivelydesignated as the first two responses of the series of six. Fouradditional responses to the continued presentation of stimuli that arevaried sequentially up or down based on the rat's response constitutethe remainder of the series.

[0080] Thus, the number of actual responses collected varied from aminimum of 4 (in the case of paw withdrawal sequentially to the firsthair, 2.0 g, descending to the weakest hair, 0.4 g: threshold lies belowthe range of actual stimuli), to a maximum of 9 (in the case of thefirst withdrawal occurring on the fifth ascending stimulis presentationat 15.1 g followed by elicitation of four additional responses, assumingthat the withdrawals continue to occur at or below 15.1 g). In caseswhere continuous positive or negative responses are observed to continueto occur to the exhaustion of the stimulis set, values of 15.00 g and0.25 g are assigned respectively. The resulting pattern of positive andnegative responses is tabulated using the convention, X=withdrawal(positive response), 0=no withdrawal (negative response), and the 50%response threshold is interpolated using the formula, 50% gramthreshold=(10[Xf=k∂])/10,000, where Xf=the value (in log units) of thefinal von Frey hair use; k=the value from the table prepared for thepattern of positive and negative responses, and; ∂=the mean difference(in log units) between stimuli.

[0081]FIG. 7 (control, n=8) shows that intrathecal administration ofBOTOX® to the neuropathic rats at a concentration of 0.625 U/kg, 0.03ng/kg (n=11), or at 3.125 U/kg, 0.15 ng/kg (n=9) clearly reduced theallodynia in rats, and that the analgesic effect lasted more than aweek. The time intervals along the x axis in FIG. 4 are time afterintrathecal administration of the BOTOX®. A higher G value indicatesthat more force is required before the paw is withdrawn.

[0082] The examples above show that intrathecal administration ofbotulinum toxin type A has a pronounced and long lasting analgesiceffect upon both inflammatory and neuropathic pain and that theanalgesic effect is dose dependent and site specific.

[0083] Additional observations showed that at the doses used intrathecalBOTOX® did not produce any significant change in blood pressure andadditionally did not cause any significant motor deficit in the subjectrats.

Example 6 Treatment of Inflammatory Pain

[0084] A patient, age 45, experiencing acute inflammatory pain istreated by intrathecal administration, for example by spinal tap to thelumbar region, with between about 0.1 U/kg and 30 U/kg of botulinumtoxin type A, the particular toxin dose and site of injection, as wellas the frequency of toxin administrations depend upon a variety offactors within the skill of the treating physician, as previously setforth. Within 1-7 days after toxin administration the patient's pain issubstantially alleviated.

[0085] The botulinum toxin can be injected at different spinal levels totreat different dermosomes, that is to treat pain in various body parts.Additionally, a catheter can be percutaneously inserted into theintrathecal space via lumbar puncture at vertebral level L3-4 or L4-5using a Tuohy needle. When CSF flow is discernible a silastic catheteris threaded cephalad using a C-arm for verification of catheterplacement. The catheter can be advanced to different vertebral locationsand/or used at different dose concentrations to treat different types ofpain and/or spasm. Thus, the catheter can be placed within theintrathecal space at the dermatomal level of the pain or spasmexperienced.

Example 7 Treatment of Neuropathic Pain

[0086] A patient, age 36, experiencing pain of neuropathic origin istreated by intrathecal administration through spinal tap to the lumbarregion of between about 0.1 U/kg and 30 U/kg of botulinum toxin type A.Within 1-7 days the pain symptoms are substantially alleviated.

Example 8 Treatment of Pain Subsequent to Spinal Cord Injury

[0087] A patient, age 39, experiencing pain subsequent to spinal cordinjury is treated by intrathecal administration, for example by spinaltap or by catheterization, to the spinal cord, such as to the lumbarregion of the spinal cord, with between about 0.1 U/kg and 30 U/kg ofbotulinum toxin type A, the particular toxin dose and site of injection,as well as the frequency of toxin administrations depend upon a varietyof factors within the skill of the treating physician, as previously setforth. Within 1-7 days after toxin administration the patient's pain issubstantially alleviated.

Example 9 Treatment of Pain Subsequent to Limb Injury

[0088] A patient, age 51, experiencing pain subsequent to injury to hishand, arm, foot or leg is treated by intrathecal administration, forexample by spinal tap or by catheterization, to the spinal cord, such asto the lumbar region of the spinal cord, with between about 0.1 U/kg and30 U/kg of botulinum toxin type A, the particular toxin dose and site ofinjection, as well as the frequency of toxin administrations depend upona variety of factors within the skill of the treating physician, aspreviously set forth. Within 1-7 days after toxin administration thepatient's pain is substantially alleviated.

Example 10 Treatment of Pain Associated With Cancer

[0089] A patient, age 63, suffering from pain associated with cancer istreated by intrathecal administration, for example by spinal tap or bycatheterization, to the spinal cord, such as to the lumbar region of thespinal cord, with between about 0.1 U/kg and 30 U/kg of botulinum toxintype A, the particular toxin dose and site of injection, as well as thefrequency of toxin administrations depend upon a variety of factorswithin the skill of the treating physician, as previously set forth.Within 1-7 days after toxin administration the patient's pain issubstantially alleviated.

Example 11 Treatment of Pain Associated With Diabetes

[0090] A patient, age 47, suffering from pain associated with diabetesis treated by intrathecal administration, for example by spinal tap orby catheterization, to the spinal cord, such as to the lumbar region ofthe spinal cord, with between about 0.1 U/kg and 30 U/kg of botulinumtoxin type A, the particular toxin dose and site of injection, as wellas the frequency of toxin administrations depend upon a variety offactors within the skill of the treating physician, as previously setforth. Within 1-7 days after toxin administration the patient's pain issubstantially alleviated.

[0091] An intraspinal neurotoxin administration method for treating painaccording to the invention disclosed herein for has many benefits andadvantages, including the following:

[0092] 1. the symptoms of pain can be dramatically reduced.

[0093] 2. the symptoms of pain can be reduced for from about two toabout four months per injection of neurotoxin.

[0094] 3. the injected neurotoxin tends to exerts a CNS site specificantinociceptive effect.

[0095] 4. the injected neurotoxin shows little or no tendency to diffuseor to be transported away from the CNS injection site.

[0096] 5. few or no significant undesirable side effects occur fromintraspinal injection of the neurotoxin.

[0097] 6. the amount of neurotoxin injected intraspinally can beconsiderably less than the amount of the same neurotoxin required byother routes of administration (i.e. intramuscular, intrasphincter, oralor parenteral) to achieve a comparable effect.

[0098] 7. The antinociceptive effects of the present methods oftenresult in the desirable side effects of greater patient mobility, a morepositive attitude, and an improved quality of life.

[0099] Although the present invention has been described in detail withregard to certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of neurotoxins can be effectively used inthe methods of the present invention. Additionally, the presentinvention includes intraspinal administration methods wherein two ormore neurotoxins, such as two or more botulinum toxins, are administeredconcurrently or consecutively. For example, botulinum toxin type A canbe administered intraspinally until a loss of clinical response orneutralizing antibodies develop, followed by administration of botulinumtoxin type E. Alternately, a combination of any two or more of thebotulinum serotypes A-G can be intraspinally administered to control theonset and duration of the desired therapeutic result. Furthermore,non-neurotoxin compounds can be intraspinally administered prior to,concurrently with or subsequent to administration of the neurotoxin toproved adjunct effect such as enhanced or a more rapid onset ofanalgesia before the neurotoxin, such as a botulinum toxin, begins toexert its analgesic effect.

[0100] Our invention also includes within its scope the use of aneurotoxin, such as a botulinum toxin, in the preparation of amedicament for the treatment of pain, by intraspinal administration ofthe neurotoxin.

[0101] Accordingly, the spirit and scope of the following claims shouldnot be limited to the descriptions of the preferred embodiments setforth above.

We claim:
 1. A method for treating pain, the method comprising the stepof intraspinal administration of a neurotoxin to a mammal, therebyalleviating pain experienced by the mammal, wherein the neurotoxin isnot attached to a neuronal targeting moiety.
 2. The method of claim 1 ,wherein the neurotoxin is a botulinum toxin.
 3. The method of claim 2 ,wherein the botulinum toxin is selected from the group consisting ofbotulinum toxin types A, B, C, D, E, F and G.
 4. The method of claim 3 ,wherein the botulinum toxin is botulinum toxin type A.
 5. The method ofclaim 2 , wherein the botulinum toxin is administered in an amount ofbetween about 10⁻³ U/kg and about 60 U/kg.
 6. The method of claim 5 ,wherein the botulinum toxin is administered in an amount of betweenabout 10⁻² U/kg and about 50 U/kg.
 7. The method of claim 6 , whereinthe botulinum toxin is administered in an amount of between about 10⁻¹U/kg and about 40 U/kg.
 8. The method of claim 7 , wherein the botulinumtoxin is administered in an amount of between about 1 U/kg and about 30U/kg.
 9. The method of claim 7 , wherein the botulinum toxin isadministered in an amount of between about 1 U/kg and about 20 U/kg. 10.The method of claim 1 , wherein the pain alleviating effect persists forup to 10 days.
 11. The method of claim 1 , wherein the pain alleviatingeffect persists for up to 20 days.
 12. The method of claim 1 , whereinthe pain alleviating effect persists for up to 3 months.
 13. The methodof claim 1 , wherein the neurotoxin is administered intrathecally. 14.The method of claim 13 , wherein the neurotoxin is administeredintrathecally to a cranial region of the central nervous system.
 15. Themethod of claim 13 , wherein the neurotoxin is administeredintrathecally to a cervical region of the central nervous system. 16.The method of claim 13 , wherein the neurotoxin is administeredintrathecally to a thoracic region of the central nervous system. 17.The method of claim 13 , wherein the neurotoxin is administeredintrathecally to a lumbar region of the central nervous system.
 18. Themethod of claim 13 , wherein the neurotoxin is administeredintrathecally to a sacral region of the central nervous system.
 19. Themethod of claim 1 , wherein the administration step includes the stepsof: (a) accessing a subarachnoid space of the central nervous system ofthe mammal, and; (b) injecting the neurotoxin into the subarachnoidspace.
 20. The method of claim 19 , wherein the accessing step iscarried out by effecting a spinal tap.
 21. The method of claim 1 ,wherein the administration step includes the steps of: (a)catheterization of a subarachnoid space of the central nervous system ofthe mammal, and; (b) injecting the neurotoxin through a catheterinserted by the catheterization step into the subarachnoid space. 22.The method of claim 21 , wherein the administration step includes, priorto the injecting step, the step of attaching to or implanting in themammal an administration means for administering the neurotoxin to thecentral nervous system of the mammal, the administration meanscomprising a reservoir of the neurotoxin, the reservoir being operablyconnected to a pump means for pumping an aliquot of the neurotoxin outof the reservoir and into an end of the catheter in the subarachnoidspace.
 23. The method of claim 1 , wherein the administration step iscarried out prior to the onset of a nociceptive event or syndromeexperienced by the mammal.
 24. The method of claim 23 , wherein theadministration step is carried out between about more than 0.5 hourbefore to about 14 days before the onset of the nociceptive event. 25.The method of claim 23 , wherein the administration step is carried outbetween about more than 0.5 hour before to about 10 days before theonset of the nociceptive event.
 26. The method of claim 23 , wherein theadministration step is carried out between about more than 0.5 hourbefore to about 7 days before the onset of the nociceptive event. 27.The method of claim 23 , wherein the administration step is carried outbetween about more than 0.5 hour before to about 4 days before the onsetof the nociceptive event.
 28. The method of claim 23 , wherein theadministration step is carried out between about more than 0.5 hourbefore to about 24 hours before the onset of the nociceptive event. 29.The method of claim 23 , wherein the administration step is carried outbetween about more than 0.5 hour before to about 6 hours before theonset of the nociceptive event.
 30. The method of claim 23 , wherein theadministration step is carried out between about 2 hours before to about5 hours before the onset of the nociceptive event.
 31. The method ofclaim 1 , wherein the administration step is carried out subsequent tothe onset of a nociceptive event experienced by the mammal.
 32. Themethod of claim 31 , wherein the nociceptive event is a neuropathic painsyndrome.
 33. The method of claim 31 , wherein the nociceptive event isinflammatory pain.
 34. The method of claim 1 , wherein the neurotoxin ismade by a Clostridial bacterium.
 35. The method of claim 34 , whereinthe neurotoxin is made by a bacterium selected from the group consistingof Clostridium botulinum, Clostridium butyricum, Clostridium beratti.36. The method of claim 1 , wherein the neurotoxin is a modifiedneurotoxin.
 37. The method of claim 36 , wherein the modified neurotoxinhas at least one of its amino acids deleted, modified or replaced, ascompared to the native neurotoxin.
 38. The method of claim 36 , whereinthe modified neurotoxin is a recombinant produced neurotoxin or aderivative or fragment thereof.
 39. A method for the in vivo attenuationof a nociceptive activity or experience of a human patient, the methodcomprising the step of intraspinal administration to a human patient atherapeutically effective amount of a botulinum toxin, thereby causingan in vivo attenuation of a nociceptive activity or experience of thehuman patient.
 40. The method of claim 39 , wherein the intraspinaladministration step is carried out subsequent to the onset of thenociceptive activity or experience.
 41. The method of claim 39 , whereinthe botulinum toxin is selected from the group consisting of botulinumtoxins A, B, C, D, E, F and G.
 42. The method of claim 41 , wherein thebotulinum toxin is botulinum toxin type A.
 43. A method for treatingpain, the method comprising the steps of: (a) selecting a neurotoxinwith antinociceptive activity; (b) choosing a portion of a centralnervous system of a patient which influences a nociceptive activity; (c)intraspinally administering to the portion of the central nervous systemchosen the neurotoxin selected.
 44. A method for treating pain, themethod comprising the step of administering a pharmaceutical preparationto the central nervous system or to a dorsal root ganglion of a mammal,thereby alleviating pain experienced by the mammal, wherein thepharmaceutical preparation comprises a neurotoxin and the pharmaceuticalpreparation is essentially free of any neuronal targeting moiety.
 45. Amethod for improving patient function, the method comprising the step ofadministering a neurotoxin to the central nervous system or to a dorsalroot gang lion of a mammal, thereby improving patient function asdetermined by improvement in one or more of the factors of reduced pain,reduced time spent in bed, increased ambulation, healthier attitude anda more varied lifestyle.